...translational applications (mechanistic studies and gene therapy treatments for neurological diseases). ...neuroscience research is the lack of a method for genetically manipulating neurons throughout the whole brain... creates a template that’s suitable for PCR amplification using pre-designed primers. By using this library...astrocytes. Why screen for IV deliverable AAVs? Typically AAVs are directly injected into the brain, but...throughout the body and is often difficult to surgically access. Therefore an IV injection is an ideal...pictures (see figure 4). It also enables single-cell anatomical study and neuronal tracing, aka determining where...

... antibiotic resistance genes so that you can specifically isolate bacteria containing your constructs ...common antibiotic resistance genes and which applications are best (or worst) for each antibiotic. The...Ampicillin Ampicillin, often just called amp, is the historical king of the antibiotic resistance realm. Amp ...immediately after it has been heat-shocked or electrically zapped. As a result, a shorter recovery period...is expensive (relative to amp), and for some applications, the benefit it offers is marginal. Fortunately...antibiotic resistance ultimately comes down to your applications and needs. The more niche and specific your ... antimicrobial agents in MIC trays. Journal of Clinical Microbiology, 23(5), 959–961. https://doi.org/...

...that, they also help us keep track of the tubes’ physical location in the lab. These barcodes additionally...  freezers depending on the sample type. It is critical for us to know which plate, rack, and freezer ...missing so that we can make a new tube from a replicate sample. The transformation After the DNA has ...plasmid can grow. As the bacteria divide, they replicate and retain the plasmid. This allows us to make...help us track each sample, but we also have a dedicated and detail-oriented lab team that works carefully...performs an average of 140 transformations. The lab typically transforms newly deposited samples from Monday...Excel sheet.3. Be specific  - When assigning a physical location to your samples, include as much detail...

...Summer Science Communication Series. We’ll be covering the basics of science communication, tips and tricks...science with others, and introducing science communication as a research field. And there will even be ...be a few tips for scientific communication (manuscripts, talks, etc…!)  Deep Dives You probably are familiar... Dives” series comes in! We’ll take a common application or concept and go beyond the basics - diving ...you’re ready for the next steps on some common applications, join us as we get into the weeds on some of...

... gives you a single level of control without modification - either Cre is there or it’s not. Cre-mediated...37 °C). Smart molecular evolution led to the identification of FLPe which has a temperature optimum of ...asymmetric target site core but its 8 bp length that is critical for Cre and FLP function. Because recombination...Recombination between the two target sites with identical sequence leads to inversion of the flanked DNA...this plasmid mix results in sparse labeling of cortical neurons and their synapses.   How can you...control   Figure 2: Expression of a morphological marker (eYFP) and synaptic marker (PSD95-mcherry...(7): 657–62. PubMed PMID: 9661200.  3. Cardin, Jessica A. et al. 2009. “Driving Fast-Spiking Cells Induces...

...parenting strategies and make important ecological indicators since species live in water and on land....protocol community! Asclepias syriaca and Asclepias curassavica (Gerog Jander) Milkweed plants are known to many... as the only Monarch butterfly food. They have chemical pathways generating interesting drug-like compounds...Lab (@EvoEmbryo) September 24, 2018   Xenopus tropicalis (Gary Gorbsky) This is not a new experimental...plants and are unusual enough to be interesting biologically. They have economic importance as harmful weeds... expensive. They are using a cool fluorescent indicator system to rapidly identify recombinants. Arthropods...

... this post I’d like to delve deeper and look specifically at the characteristics and capabilities an ELN...facilitate true reproducibility. The following are critical: (1) connectivity to other data sources used in...likely stored in institutional and lab servers specifically designed to hold large amounts of data. Although...group’s research – it makes no sense and is not practical to move all this data in bulk into a lightweight...lightweight tool like an ELN. It is nevertheless critically important to be able to conveniently reference specific...with a particular experiment or project thus significantly enhances the ability of others to subsequently... both accept deposit of data associated with publications into one of five general purpose open source...

...expression limiting regulatory elements or use a non-canonical code (Gustafsson et al., 2004), such as Hoogsten...folding and stability, change post-translational modification sites and even affect protein function (Mauro...recognize multiple synonymous codons) may actually be critical for determining the rate of translation. The ribosome...10.1101/gr.139758.112 Mauro VP, Chappell SA (2014) A critical analysis of codon optimization in human therapeutics...synonymous codon usage bias, and its potential applications. Nucl Acids Res 15:1281–1295 . https://doi.org... GluCl ion channel genes for mammalian cells dramatically improves expression levels. Journal of Neuroscience...synthetic? How Addgene;s dataset reveals trends in biological innovation Resources on Addgene.org Check out...

...described above showed successful target modification, indicating that AAV is a good delivery vehicle for...overall packaging capacity but necessitating purification and co-infection of two AAVs. Cas9 orthologs...post-injection. Livers from these mice were histologically normal and liver injury markers were not increased...the AAV-SaCas9-sgRNA constructs mediate genome modification, but they did so without a substantial immune...enough to deliver with AAV. This system fuses catalytically dead dCas13b to the catalytic domain of RNA ...finding smaller Cas9 orthologs works for some applications, it doesn’t eliminate the need to deliver larger...

...very little data on which might work in your application. It can be quite difficult to decide on which...be searched and sorted by gene/target, name, application, pass/fail rating, sample species, antibody species... how the antibody performed in the submitted application, along with a brief description of the experiment...experiment. If that data was included in a publication, you'll find that information in the report as well... section will vary slightly depending on the application — flow cytometry, western blot, immunohistochemistry...data showing how the antibody worked in your application. The submission process is easy, with simple,...

...window onto communication between the muscle and brain, which may have therapeutic implications, say Hyo ...metabolites that are dynamically secreted by muscle during exercise to meet the brain’s physiological demands,” ...protects the brain from depression. Mice that are genetically modified to produce extra PGC-1a1 only in their..., the brain is protected from stress-induced biochemical changes that could otherwise lead to depression...

...speak for you (and you for them) Communication Being able to communicate effectively is part of what makes...excellent Technical knowledge and creativity Become a “thought leader,” develop a technical “niche,” develop...productive. Take all the opportunities you can to be communicating about science with others. If opportunities ... (be on a speaker committee) Follow industry publications like FierceBiotech to “learn the business” Attend...

...clones, it’s wise to quickly assess whether a significant number of cells have been edited. For many experiments...enzyme screening for resistivity to digestion, indicating targeted edits.   Interested in this method...sequencing confirmation. All knock-ins A method applicable to knock-ins both large and small is a software...consider using a version of Cas9 that has been specifically engineered for reduced off target editing, such...Tao Chen, Jos Jonkers, Bas van Steensel. Easy quantification of template-directed CRISPR-Cas9 editing. (...and variant-aware in silico off-target site identification for CRISPR genome editing. (2019) Bioinformatics...Resources at Addgene: Read our CRISPRGuide Get practical help from our CRISPR Protocols Find gRNA Design...

...which to test the validity of your biological model and the technical performance of your assay. But, in... a more-typical scale of tissue culture while still offering the potential for true biological insight...components, the model system and the assay are just as critical for success. Sometimes, you might have a few positive...facilitating more rapid follow-up. We hope the biological community will find these reagents to be useful...

...al. "Systematic identification of signaling pathways with potential to confer anticancer drug resistance... therapies,” said Kris Wood of Duke University Medical Center. “While we do not yet have a comprehensive... of 17 signaling pathways that are frequently implicated in cancer cell proliferation, survival, differentiation... make the library usable for pooled screens. Applications of the cancer pathways ORFs kit Wood and his... he said. “It could [be used to screen for] morphological features, migration, or survival in different...

...Thousands of mutations have been implicated in cancer. Yet, the majority of them remain uncharacterized...annotate genetic perturbations that may play significant roles in cancer pathogenesis. Their work culminated... of mutations they identified included gene amplifications and fusions, missense mutations, and indels...mutations were found to be “neutral,” having no significant effect on cell viability compared to a wild-type...Approximately ~22% of the mutations, however, significantly increased cell viability, and these “activators...aberration drivers of pancreatic cancer." Nature communications 7 (2016): 10500. PubMed PMID: 26806015. PubMed...

...with the gRNA), application, cas9 species, PubMed ID, and depositor. The CRISPR applications we currently...shown to work in CRISPR experiments. Addgene’s dedication to resource sharing has led us to develop a searchable...validated gRNA Target Sequence Table. Disclaimer: The efficacy of gRNAs is impacted by the target location, target...the carats in the datatable headers to sort alphabetically or in ascending or descending order, in the...and final experimental outcome in the original publication. As you develop and confirm new gRNAs, please...

...Since the first research applications of GFP were published in the 1990s, biologists have spent a lot... mammalian cells, and it may find wide use in biological research. Photoconvertible fluorescent proteins...color was observed after excitation at 488 nm, indicating that spontaneous conversion did not occur. Tobacco... Sattarzadeh et al., Sci Rep. 2015 CC BY 4.0 Applications and limitations of GFP photoconversion One concern...2):131-3.  PubMed. Find plasmids from this publication at Addgene. Subach OM, Patterson GH, Ting LM...nmeth.1664. PubMed. Find plasmids from this publication at Addgene.   Addgene’s Fluorescent Protein...

...scientific community support open sharing. But, one critical part was missing when the COVID-19 plasmids first...Carolina at Chapel Hill University of Illinois Chicago                     Boston Children's Hospital                           ...                           National Institute for Physiological Sciences Broad Institute                                       ...                                       Chinese Academy of Medical Sciences University of Texas at Austin University of California...part of research projects, another way is to systematically offer thousands of constructs for tagged and...enables research on solute carriers (SLCs) for medical research Recently, the Michael Davidson Fluorescent...

... QC/QA team as a DNA Sequencing Intern. The application process When I first saw the comprehensive job...immediately clear that Addgene wanted to ensure applicants understood what the expectations were for the...the intended position. After submitting my application, I was contacted to participate in a brief fifteen...the depositing labs intended. With the use of applications like SnapGene and Geneious, I was able to determine...team and shows how they fit into the system. Specifically as a co-op, it is encouraged to explore different...currently working on projects for the Science Communication function at Addgene. Working a 9-5 As it turned...

...technology, the Vale and Weissman labs took this biological lesson and created a system to amplify fluorescent...modified the GCN4 sequence to increase its alpha-helical structure and stability, creating v4 SunTag. Since...aggregation, it’s recommended for most imaging applications. What can you do with SunTag? Tanenbaum et al...flames,” it’s clear that the system is broadly applicable across many areas of research. Beyond fluorescence...activation of target genes, but we’ll save that application for another day! References 1. Tanenbaum, Marvin...et al. “A protein-tagging system for signal amplification in gene expression and fluorescence imaging....

...generally linked to dopamine (DA), a well-known reward chemical and neurotransmitter. But aside from DA’s famous...at a certain frequency, and this frequency can indicate behavioral state, for example, if someone is awake... it can be performed in a live awake animal by surgical implantation of the probe into a specific brain...in freely moving mice Record DA dynamics of optogenetically activated and inhibited neurons in live mice...neuronal imaging of dopamine dynamics with designed genetically encoded sensors." Science 360.6396 (2018): eaat4422... PMCID: PMC6287765. Sun, Fangmiao, et al. "A genetically encoded fluorescent sensor enables rapid and ...

...swimming where they periodically beat their tails left and right in a symmetrical motion to move forward...behavioral studies. In neuroscience, opsins are typically ion channels that allow charged particles to cross...Kolmer-Agduhr cells was sufficient to trigger the symmetrical tail movement unique to spontaneous slow forward... photostimulation in order to drive the full symmetrical response.  The function of Kolmer-Agduhr cells...Aristides B., Filippo Del Bene, and Herwig Baier. "Optical control of zebrafish behavior with halorhodopsin...

... (e.g. 6xHis containing pDEST-HisMBP). After purification the protein can be released from the resin and...types of yeast expression vectors. Consider your applications and experimental design. You can create a new...more traditional approach of an autonomously replicating vector that carries either a wild-type version...expression pattern of a defined promoter region, ectopically expressing YGOI in a pattern that allows the ...a variety of other types of vectors for most applications used to study gene function. From making transgenic...transgenic animals, gene targeting and genome modifications to reporter vectors (e.g. pBV-Luc) and gene ...

... Foundation Round Table. In any field, good communication skills help when it comes to finding a job. ...an argument for taking the time to hone your communication skills. So, where can a graduate student or ...opportunities than ever before for scientists to communicate about their work and their interest in science...your area or at your institution; the news or communications office is a good place to start. It’s not unusual...about science writing as a career, the Science Communication Program at the University of California, Santa... your research stories. Consider practicing communicating your research by guest blogging here, just leave...

...problems, develop general technical proficiency, communicate your work, maintain ethical standards and interact...? Listen to our podcast interview with Harvard Medical School researcher Connie Cepko to learn about her... you admire her management style? Does he have ethical behavior? Does the lab encourage diversity? Knowing...websites listing alumi and it is easy to use publication records, university websites, ResearchGate and...

...appreciable levels of modification. Whole-genome sequencing of cells modified by CRISPR indicates that the consequences...20nt-NGG] and [5’-CCN-20nt], as it is equally efficacious to target the coding or non-coding strand of ...desired location of edit; efficiency decreases dramatically when the cut site is >30nt from the proximal... via CRISPR cutting. For CRISPRa, it is most-efficacious to target a ~100nt window upstream of the transcription...sequence may not be available. Predicting gRNA efficacy We and others have examined the ability to use...needs to test in the lab. Importantly, for any modification of interest, it would be unwise to make conclusions...

...natural and engineered repressible promoters. Chemically repressible promoters Tet-Off In its native context... GAL80 under different promoters and create sophisticated patterns of UAS-driven gene expression. To further... PMCID: PMC2942869. Find plasmids from this publication at Addgene. del Valle Rodriguez, Alberto, Dominic... PMCID: PMC2883883. Find plasmids from this publication at Addgene. Wei, Xing, Christopher J. Potter... PMCID: PMC3846601. Find plasmids from this publication at Addgene. Riabinina, et al. “Improved and ... PMCID: PMC4344399. Find plasmids from this publication at Addgene. Riabinina O, Potter CJ. The Q-System...expression of genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res...

...the gRNA design process and rules for various applications is necessary going forward. The standardization...benefits to researchers conducting basic or early pre-clinical research. However, standard terms and techniques... I think I may have finally grasped just how complicated DNA repair pathways are. I fully admit to being...to me. But coming to #KSgenome and seeing the intricacies of DNA repair has emphasized how essential these...Alt-NHEJ), has been harnessed for CRISPR knock-in applications. CRISPR 101: Targeting RNA with Cas13a (C2c2...for CRISPR Genome Editing Other resources and publications CRISPOR gRNA design tool Researchers at the ...

...supercoiled DNA ends to facilitate replication. The TOPO technique specifically uses Vaccinia virus-isolated...insert comes from using Taq polymerase for the amplification step since Taq polymerase leaves a single deoxyadenosine...minutes (incubating the full 30 minutes will not significantly improve the transformation efficiency).  5. ... has an error rate of about 1 in 3,500 bases. Typically polymerases with proofreading functionality are...are planning on transforming your reaction into chemically or electro-competent E. coli - excess salt causes...

...University and Medical Center  Weizmann Institute of Science Whitehead Institute for Biomedical Research ...time of request MTAs and deposit agreements to basically instantaneous in many cases.  Suggest that they...your organization below? Don’t worry--we check periodically for possible new awardees, and we’re happy to...University Rockefeller University  Salk Institute for Biological Studies The Scripps Research Institute St. Jude...Massachusetts, Worcester  University of Nebraska Medical Center University of Pennsylvania  University of...

...Addgene! Archon1, a voltage sensor AAV Historically, electrical activity in the brain was measured with...kinetics) that makes them suitable for different applications. We recently released a new variant of jGCaMP7... been developed and are suited for different applications. The iGABASnFR GABA sensor (pAAV.hSyn-FLEX.iGABASnFR...voltage reporter allows a way to monitor neuronal electrical activity through imaging. Since it’s illuminated...Also coming in our new 20 ul trial size! Voltage indicators Plasmid Serotype Name 119036  AAV1  pAAV-hsyn-flex-Voltron-ST...

...infection, because this setup allows for experimental replicates for each of the gRNAs. The idea was to assign...genetic tool for labeling just a fraction of a genetically-defined subset of cells. By labeling fewer cells...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ...measurements, or monitoring in vivo. To overcome these technical issues, Sabrina Büttner’s lab designed a calcineurin... reporter (blue) and stained with PI (pink) to indicate cell death. Image from Diessl et al., 2020. ...protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry. In comparison...

... (AmpR) and origins of replication (ori and f1 ori) which allow it to replicate.   How can I prevent...the bacterial selection marker and origin of replication. During bacterial culture, only the plasmid with...the antibiotic resistance gene and origin will replicate, and the transgene will be lost. Over time, bacteria...the recombined plasmid is still visible. This indicates that the DNA prep contains a mixture of the full...MK, Rudolph C (2019) Segmented poly(A) tails significantly reduce recombination of plasmid DNA without ...

... “species on species”) immunochemistry (IHC) applications an issue? The culprits are endogenous IgGs in...IgGs and your primary antibody, resulting in significant background staining (Figure 1A). Not all tissues...Figure 2B) Some labs have found that you can significantly decrease background staining by mixing your ...Randolph-Habecker J (2013). A Flexible Mouse-On-Mouse Immunohistochemical Staining Technique Adaptable to Biotin-Free...Partridge TA (1998). A New Blocking Method for Application of Murine Monoclonal Antibody to Mouse Tissue...Tuson JR, Pascoe EW, Jacob DA (1990) A novel immunohistochemical technique for demonstration of specific binding...

...lab used an alternative strategy to develop a genetically encoded antibody based probe that detects the...antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based...National Academy of Sciences of the United States of America, 69(9), 2659–2662. PubMed PMID:4560694. PubMed ... and antibodies with crystalline papain. The Biochemical Journal, 73(1948), 119–126. PubMed PMID: 14434282..., T., Sato, Y., … Stasevich, T. J. (2019). A genetically encoded probe for imaging nascent and mature ...mature HA-tagged proteins in vivo. Nature Communications, 10(1). PubMed PMID: 31270320. PubMed Central PMCID...

...Harvard Medical School and runs everything science (CSO). Clinton is a cost analyst at Dow Chemical (a Fortune...a company in the health sciences without those critical three letters after your name (P-H-D).  Finally...two projects, including one led by a talented biomedical engineering team from MIT.   I would like to ...launching a company without partners.  You need a dedicated team that you know well and trust.  This will ...majority of these meetings acted as a platform for significant company advancement. Be relentless The world ...

...databases available, Web of Science. For each publication, Web of Science provides basic bibliographic ...Microbiology, Biochemistry & Molecular Biology, and Biochemical Research Methods. We also pulled the big interdisciplinary...Science, PLOS One, and PNAS. We then broke the publications from these journals into two groups: those containing...the papers with plasmids deposited at Addgene – typically about four times as many as papers without plasmids...differences in citations due to the journal of publication. Table 1 provides an example calculation of the...Addgene in them. The adjustment is done within publication years. What do we find once we do this adjustment...

...field has exploded so quickly, citing the first clinical trial with iPSCs with his collaborator Takahashi... have profound impacts on the development of applications with the potential to save lives. While most...picture perspective: Dr. Yamanaka talked about his clinical trial patients with advanced macular degeneration... ground-breaking work on spinal cord injuries; Jessica Young shared research on stem cells and Alzheimer's...Eggan presented work that is leading to promising clinical trials for ALS.   I have an uncle with Parkinson's...the first stem cell line that could be used in clinical trials. The quality control process alone takes...

...The catalytically dead Cas9 protein (dCas9) is well known for its ability to bind DNA targets without...has been widely adapted for a wide variety of applications: base editing, CRISPR activation and inhibition...Pederson’s lab at the University of Massachusetts Medical School developed a dCas9-based multicolor labeling...RFP) to the genomic loci of interest. Pairs of identical hairpins within one gRNA will result in a blue...National Academy of Sciences of the United States of America 112 10 (2015): 3002-7. PubMed PMID: 25713381 PubMed...al. “CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging.” Nature Methods 15 (2018)...